During the past 15 years, we have carried out several different systematic screens for small regulatory RNA genes in E. coli. These screens, which have included computational screens for conservation of intergenic regions and direct detection after size selection or co-immunoprecipitation with the RNA binding protein Hfq, are all applicable to other organisms. We are now examining small RNA expression using deep sequencing to further extend our identification of small RNAs, particularly antisense RNAs. A large focus of the group has been to elucidate the functions of the small RNAs that we and others have identified. Early on we showed that the OxyS RNA, whose expression is induced in response to oxidative stress, acts to repress translation through limited base pairing with target mRNAs. We discovered that the OxyS action is dependent on the Sm-like Hfq protein, which functions as a chaperone to facilitate OxyS RNA base pairing with its target mRNAs. More recently we carried out an extensive mutational study of Hfq (1). This analysis revealed that amino acids on three different RNA interaction surfaces--the proximal face, the distal face and the rim of the doughnut-shaped protein--differentially impact Hfq association with small RNAs and their mRNA targets. It is now clear that Hfq-binding small RNAs, which act through limited base pairing, are integral to many different stress responses in E. coli. We showed that FnrS, whose expression is induced by FNR upon a shift from aerobic to anaerobic conditions, acts to down regulate the levels of a variety of mRNAs encoding metabolic enzymes not needed under anaerobic conditions. We also reported that the Spot 42 RNA, whose levels are highest when glucose is present, plays a broad role in catabolite repression by directly repressing genes involved in central and secondary metabolism, redox balancing, and the consumption of diverse nonpreferred carbon sources (2). Many of the genes repressed by Spot 42 are transcriptionally activated by the global regulator CRP. Since CRP represses Spot 42, these regulators participate in a specific regulatory circuit called a multioutput feedforward loop. We found that this loop can reduce leaky expression of target genes in the presence of glucose and can maintain repression of target genes under changing nutrient conditions. In more recent work, we discovered that the McaS RNA, whose levels are elevated in stationary phase or when glucose is limiting, regulates mRNA targets involved in various aspects of biofilm formation (3). McaS represses csgD, the transcription regulator of curli biogenesis and activates flhD, the master transcription regulator of flagella synthesis leading to increased motility, a process not previously reported to be regulated by sRNAs. McaS also regulates pgaA, a porin required for the export of the polysaccharide poly &#946;-1,6-N-acetyl-d-glucosamine. Consequently, high levels of McaS result in increased biofilm formation while a strain lacking McaS shows reduced biofilm formation. Based on these observations, we propose that McaS modulates steps in the progression to a sessile lifestyle. While we assumed that sRNAs act solely by one mechanism, to our surprise, we discovered that McaS acts by two different mechanisms: base-pairing and protein titration (4). McaS base pairs with the csgD and flhD mRNAs, respectively, resulting in down-regulation and up-regulation of the corresponding cell surface structures. In contrast, McaS activates pgaA by binding the global RNA-binding protein CsrA, a negative regulator of pgaA translation. The McaS RNA bears at least two CsrA-binding sequences, and inactivation of these sites compromises CsrA binding, PGA regulation, and biofilm formation. Moreover, ectopic McaS expression leads to induction of two additional CsrA-repressed genes encoding diguanylate cyclases. Thus McaS is a dual-function sRNA with roles in the two major post-transcriptional regulons controlled by the RNA-binding proteins Hfq and CsrA. In addition to small RNAs that act via limited base pairing, we have been interested in small antisense RNAs that have the potential to form extensive base pairing interactions with their mRNA targets encoded on the opposite strand. We showed that base pairing of GadY encoded antisense to the gadXW mRNA results in processing giving rise to two halves that accumulate to higher levels than the full length mRNA. Multiple enzymes, including the double strand RNA-specific endoribonuclease RNase III, are involved in the GadY-direct cleavage. We also reported that a large class of antisense RNAs acts to repress the synthesis of small toxic proteins. For example, in characterizing the Sib RNAs, which are encoded by five repeats in E. coli K-12, we observed an overexpression phenotype reminiscent of plasmid addiction. Further examination of the SIB repeat sequences revealed conserved open reading frames encoding highly hydrophobic 18-19 amino acid proteins (Ibs) opposite each sib gene. The Ibs proteins were found to be toxic when overexpressed, and this toxicity could be prevented by co-expression of the corresponding Sib RNA. Computational screens together with experimental validation have shown that these small hydrophobic protein-antisense RNA gene modules, termed type 1 toxin-antitoxin modules, are much more widely distributed among bacteria than previously appreciated. Finally, we discovered the an abundant and broadly-conserved small RNA mimics the DNA structure of an open promoter and modulates RNA polymerase activity. Studies to further characterize other Hfq-binding RNAs and antisense RNAs and to elucidate the roles of small RNAs that act in ways other than base pairing are ongoing.